Fluorescent dyes have a wide variety of uses including the labeling of antibodies, DNA, carbohydrates and cells. In order for a fluorescent dye to function as a label, the dye must bind to the molecule or cell to be labeled. Fluorescent labels are therefore designed to include at least one reactive moiety which reacts with amino, hydroxyl and/or sulfhydryl nucleophiles present on the molecules being labeled. Examples of suitable reactive moieties include carboxylic acids, acid halides, sulfonic acids, esters, aldehydes, disulfides, isothiocyanates, isocyanates, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridines, mono- or di-halogen substituted diazines, maleimide, aziridines, sulfonyl halides, hydroxysuccinimide esters, hydroxysulfosuccinimide esters, imido esters, hydrazines, azidonitrophenyl, azides, 3-(2-pyridyl dithio)-propionamide and glyoxal. Additional suitable reactive moieties for use in fluorescent labels are described in U.S. Pat. No. 5,268,486 which is incorporated herein by reference.
Fluorescent dyes commonly have an absorbance range of between about 300 and 900 nm and preferably have a Stokes shift of at least about 20 nm. Fluorescent dyes that absorb in the 500 to 900 nm range are preferred because they are spectrally removed from other components that may be present in a biological sample and because they may be used with inexpensive light sources. Fluorescent dyes that have a high extinction coefficient and a high quantum yield are also preferred.
Fluorescent dyes used for labeling biomolecules, such as carbohydrates, proteins and DNA, are preferably water soluble since the biomolecules to be labeled generally have limited solubility in nonaqueous solvents. It is known to increase the water solubility of a dye by adding hydrophilic groups such as sulfonate groups and hydroxyl groups. Examples of water soluble dyes that may be used as fluorescent probes include fluorescein (Coons, et al., J. Exp. Med. (1950) 91 1-13), phycobiliproteins (Oi, et al., J. Cell. Biol. (1982) 93 981) and Cy5 (Mujumdar, et al., Bioconjugate Chem. (1993) 4 105-111).
It is important that the fluorescent dye is photostable. However, dyes with a fluoresence absorbance greater than 500 nm tend to be less photostabile.
Fluorescent dyes also should not be prone to aggregation. Dye aggregation, also known as "stacking" increases the frequency of fluorescence quenching which reduces the strength of the fluoresence signal observed. Most fluorescent dyes are large planar molecules, are intrinsically hydrophobic and therefore have a tendency to aggregate or "stack," especially in aqueous solutions. Dyes with a fluoresence absorbance greater than 500 nm generally have a greater tendency to stack due to their increased size and associated lower solubility. Non-aggregating, photostable fluorescent dyes with a fluoresence absorbance greater than 500 nm are therefore needed.
Fluorescent probes are particularly prone to stack in high salt solutions and when in high local concentrations on protein surfaces. For example, tetramethylrhodamine, a commonly used laser dye, produces protein-dye conjugates which predominantly consist of the aggregated dye. Aggregated dyes appear blue-shifted by visible absorbance spectra. Amino-substituted cyanine dyes, such as IR144, are prone to aggregation in aqueous solutions, even in low-salt solutions (i.e. 0.1M NaCl). Non-aggregated amino-substituted cyanine dyes have only been found to exist in organic solvents. The absorbance spectra of protein-dye conjugates can be simulated by obtaining spectra of the dye in high salt solutions (e.g. 4M NaCl). Dye aggregation may be minimized by constructing highly ionic dyes such as arylsulfonates taught in U.S. Pat. No. 5,268,486 or by using naturally occurring fluorescent probes such as phycobiliproteins.